It was previously shown that arbuscular mycorrhizal
fungi (AMF) exert a significant improvement of growth in
a tolerant white poplar (Populus alba L.) clone (AL35) grown
on Cu- and Zn-polluted soil via foliar alterations in the levels
of defence/stress-related transcripts and molecules. However,
nothing is known about the epigenetic changes which occur
during tolerance acquisition in response to heavy metals
(HMs) in the same mycorrhizal vs. non-mycorrhizal poplar
plants. In order to analyse the epigenome in leaves of AL35
plants inoculated or not withAMF and grown in a greenhouse
on multimetal polluted or unpolluted soil, the Methylation
Sensitive Amplification Polymorphism (MSAP) approach
was adopted to detect cytosine DNA methylation. Modest
changes in cytosine methylation patterns were detected at first
sampling (4 months from planting), whereas extensive alterations
(hypomethylation) occurred at second sampling (after
6 months) in mycorrhizal plants grown in the presence of
HMs. The sequencing of MSAP fragments led to the identification of genes belonging to several Gene Ontology
categories. Seven MSAP fragments, selected on the basis of
DNA methylation status in treated vs control AL35 leaves at
the end of the experiment, were analysed for their transcript
levels by means of qRT-PCR. Gene expression varied in
treated samples relative to controls in response to HMs and/
or AMF inoculation; in particular, transcripts of genes involved
in RNA processing, cell wall and amino acid metabolism
were upregulated in the presence ofAMF with or without
HMs.

It was previously shown that arbuscular mycorrhizal
fungi (AMF) exert a significant improvement of growth in
a tolerant white poplar (Populus alba L.) clone (AL35) grown
on Cu- and Zn-polluted soil via foliar alterations in the levels
of defence/stress-related transcripts and molecules. However,
nothing is known about the epigenetic changes which occur
during tolerance acquisition in response to heavy metals
(HMs) in the same mycorrhizal vs. non-mycorrhizal poplar
plants. In order to analyse the epigenome in leaves of AL35
plants inoculated or not withAMF and grown in a greenhouse
on multimetal polluted or unpolluted soil, the Methylation
Sensitive Amplification Polymorphism (MSAP) approach
was adopted to detect cytosine DNA methylation. Modest
changes in cytosine methylation patterns were detected at first
sampling (4 months from planting), whereas extensive alterations
(hypomethylation) occurred at second sampling (after
6 months) in mycorrhizal plants grown in the presence of
HMs. The sequencing of MSAP fragments led to the identification of genes belonging to several Gene Ontology
categories. Seven MSAP fragments, selected on the basis of
DNA methylation status in treated vs control AL35 leaves at
the end of the experiment, were analysed for their transcript
levels by means of qRT-PCR. Gene expression varied in
treated samples relative to controls in response to HMs and/
or AMF inoculation; in particular, transcripts of genes involved
in RNA processing, cell wall and amino acid metabolism
were upregulated in the presence ofAMF with or without
HMs.